Detection of a mutation in a DNA fragment is important for many reasons, for example determining the existence of disease, malfunction or abnormality in an animal or human organism. By way of background, DeoxyriboNucleicAcid (DNA) is the chemical inside the nucleus of a cell that carries the genetic instructions, or code, for making living organisms.
DNA comprises two long, molecular strands lying side by side in a double spiral referred to as a double helix. The double helix structural arrangement of DNA looks something like a long ladder twisted into a coil. The sides of the "ladder" are formed by sugar and phosphate molecules, and the "rungs" are made of a large number of chemical building-blocks called bases, with a pair of bases forming the rung. The bases are referred to by the code letters A, T, G and C which stand for the chemicals adenine, thymine, guanine, and cytosine, respectively. At each end of a rung are complementary bases (i.e. the "base pair") which form the bond between the two strands forming the DNA. In base pairing, adenine (A) always pairs with thymine (T), and guanine (G) always pairs with cytosine (C). That is, if on one side of the ladder rung there is an A, the base on the other side of the ladder rung is a T, and if one end of the rung is a G, the opposite end is a C. As a result, the two strands of the DNA structure are inverted copies of each other.
Although there are only four different kinds of bases, the order in which they appear along the strand of DNA dictate the genetic code of an organism. A DNA strand can therefore be referred to by its unique sequence of letters commonly called a DNA sequence. The effort of determining the exact order of the base pairs in a segment of DNA is referred to as DNA sequencing.
The strength of the bond between the two strands of the DNA structure is subject to several factors. One factor is the length of the DNA fragment. Longer fragments possess a stronger bond between the strands than shorter fragments. In addition, the specific sequence order of the fragment has an effect on the strength of the bond. That is, a strand sequence of GGGAAATTT has a different bond strength than a strand sequence of GATGATGAT. A third factor in bond strength is the actual bond pair itself. Bonds formed between Gs and Cs are stronger than bonds formed between As and Ts
A mutation is a permanent structural alteration in DNA. These mutations in some cases may have no effect on the organism however, in some instances they may cause disease, malfunction or abnormality in the organism. Accordingly, it is beneficial to identify such mutations as well as to determine whether an identified mutation exists in a particular individual.
A method for determining the existence of a mutation has evolved where a DNA fragment is exposed, for a period of time, to a threshold temperature, specific to that DNA fragment, which forces the two strands of the DNA apart, breaking the bond between the base pairs. The percent of the strand pair which is split apart at different moments in time, is referred to as the percent melted (or fraction single stranded).
A copy of a "good" (mutation-free) DNA fragment is typically made using a process called polymerase chain reaction (PCR). This "good" DNA fragment is separated and each strand is annealed with a strand from a DNA fragment which includes a mutation, the strands of which have also been separated. As mentioned above, the two strands of a DNA structure should be inverted copies of each other. When the series of bases on one copy of the DNA fragment do not match the series of bases on the other copy of the pair, a "mutation" is said to exist. Where a mutation in the DNA structure exists, the bond between the two strands is weakened. In the instance where a mutation exists within a strand, the percent melted at a specific period of time will differ from that if the strand had no mutations. A number of analytic separation methods can be used to detect differences in double stranded character of DNA.
The threshold temperature for analyzing a given DNA fragment for mutations is dependent on the length of the fragment as well as the specific fragment sequence. As described above, the length, base pairing and sequence order of the DNA fragment are each determinative of the strength of the bond and are, therefore, factors in determining the amount of heat required to separate the strands of the DNA fragment.
Historically, more is known about the separation of DNA fragments than is known about specific mutations. The separation of DNA fragments using chromatographic techniques has been studied and reported on by Peter Oefner and colleagues. See "High Resolution Liquid Chromatography of DNA Fragments on Non-Porous Poly(styrene-divinylbenzene) Particles", Nucleic Acids Research, Volume 21, No. 5, pages 1061-1066 (1993). In U.S. Pat. No. 5,585,236 to Bonn et al., an improved chromatographic technique for separating nucleic acids is disclosed. More recently, Oefner disclosed the exploitation of the chromatographic separation technique for mutation detection in a "DHPLC Workshop" at Stanford University, California, Mar. 17, 1997. By that time, the chromatographic technique was referred to and known as DHPLC (Denaturing High Performance Liquid Chromatography). A specific example of the DHPLC technique for detection of Y chromosomes in biallelic polymorphisms is given by Peter A. Underhill et al., "Detection of Numerous Y Chromosomes Biallelic Polymorphisms by Denaturing High Performance Liquid Chromatography", Genome Research 7:996-1005 (1997). Also see, Oefner, P. J. and Underhill, P. A. U.S. Pat. application, Ser. No. 08/512,681, "Detection of DNA Heteroduplex Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing", filed on Aug. 8th, 1995.
Briefly summarizing the DHPLC method for separating DNA strands, a straw-like apparatus, referred to as a separation column, is adjusted to a specific concentration of acetonitrile solution and a specific temperature. A DNA fragment is prepared from a given individual, typically using PCR which provides multiple copies of a piece of DNA. The DNA fragments are injected onto the column and adhere. The concentration of the acetonitrile solution is gradually increased and the elution rate of the DNA fragments is monitored. Typically, the process is repeated for the same fragment from multiple individuals and the patterns of elution of the DNA fragments over time are compared. For those individuals whose DNA includes a mutation, a lower concentration of acetonitrile will cause an amount of the DNA to strip from the column than for those individuals whose DNA samples do not include a mutation.
Accordingly, in the various foregoing disclosures of the DHPLC technique, there are two conditions which must be specified by the user in order to analyze any specific DNA fragment: (1) temperature and (2) percent acetonitrile, which is the solution used by the chromatography column. In the current publications, the key or threshold operating temperature used for the chromatography columns is assumed as a given value or, at most, described as being empirically determined. That is, multiple DNA test fragments are studied under a range of operating temperatures. After much experimentation, a viable temperature is determined. See page 1003, right hand column of Underhill, et al. Such empirical determination is often inexact, time consuming and laborious.